Antifungal Activity and Mechanism of the Essential Oils from Litsea
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Antifungal activity and mechanism of the essential oils from Litsea (Litsea cubeba), Melissa (Melissa ocinalis), Palmarosa (Cymbopogon martini) and Verbena (Verbena ocinalis) and their major active constituents against Trametes hirsuta and Laetiporus sulphureus Yongjian Xie ( [email protected] ) Zhejiang A & F University https://orcid.org/0000-0001-8225-7720 Xi Yang Zhejiang A&F University Hui Han Zhejiang A&F University Zhilin Zhang Hubei Engineering University Dayu Zhang Zhejiang A&F University Research Article Keywords: Antifungal activity, Essential oils, Litsea cubeba, Geranial, Membrane damage Posted Date: August 13th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-680348/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/24 Abstract Antifungal activities of 37 essential oils (EOs) against two wood-decaying fungi, Trametes hirsuta and Laetiporus sulphureus were screened in vitro, and investigated the underlying mechanism. Of the 37 EOs, litsea (Litsea cubeba), melissa (Melissa ocinalis), palmarosa (Cymbopogon martini), and verbena (Verbena ocinalis) demonstrated strong antifungal activity, in which litsea oil exhibited the strongest antifungal property against T. hirsuta and L. sulphureus, with IC50 values of 72.3 and 40.2 µg/ml, respectively. The compositions of litsea, melissa, palmarosa, and verbena EOs were analyzed using a gas chromatography-mass spectrometry method and demonstrated geranial, geraniol, neral, and citral as their major active constituents. Among of them geranial exhibited the strongest antifungal activity against T. hirsuta and L. sulphureus, with IC50 values of 56.6 and 33.3 µg/ml, respectively. These EOs and their major active constituents increased the plasma membrane permeability of T. hirsuta and L. sulphureus, resulting in the leakage of nucleic acid, protein, and soluble sugar. Results indicate that the EOs of litsea, melissa, palmarosa, and verbena and its major constituents inhibited T. hirsuta and L. sulphureus growth by targeting its plasma membrane. 1. Introduction The biodegradation of lignocellulosic materials occurs by beetles, fungi, marine borers, and termites; among these, fungi are recognized to cause the greatest nancial loss of wooden products (Wu et al. 2012). Decay fungi, molds, and stain fungi are universally recognized as major wood-degradation fungi (Bakar et al. 2013). In general, traditional wood preservatives are ammoniacal copper quaternary (ACQ), chromated copper arsenates (CCAs), copper azole (CA), and copper (II) dimethyldithiocarbamate (CDDC), which signicantly affect human health and the environment (Chen et al. 2014). Therefore, there is an urgent need to research and explore more eco-friendly, convenient, and highly effective benign wood preservatives for lignocellulosic materials. In recent years, many natural plant products that are non-residual, biodegradable, and environmentally friendly have been shown to be excellent potential alternatives for preserving the wood industry (Xie et al. 2017a). Studies have shown that plant essential oils (EOs) from Calocedrus fromosana, Cryptomeria japonica, Cinnamomum osmophloem, Ci. zeylanicum, Cymbopogon citratus, Cunninghamia konishii, Eucalyptus camaldulensis, Eugenia caryophyllata, Machilus philippinensis, Origanum vulgare, Pelargonium graveolens, and Thymus vulgaris have antifungal properties (Cheng et al. 2005, 2006, 2011; Ho et al. 2010; Xie et al. 2015, 2017a; Salem et al. 2016). As is well known, the strong antifungal activity of EOs against wood decaying fungi were contributed to a rich of monoterpenes, sesquiterpenes and phenylpropanoids (Cheng et al. 2012; Zhang et al. 2016a). Most EOs and their compounds destroy the integrity of fungal cell membranes, resulted in the outow of intracellular components and cell death (Kalily et al. 2016; Zhang et al. 2016b; Zhou et al. 2017; Souza et al. 2020; Yan et al. 2020). Research on EOs as biological agents for protecting wood and prolonging their application life and as an alternative to chemical wood preservatives is becoming increasingly a necessity. For this reason, this Page 2/24 study examined the antifungal activity of 37 EOs against wood-decay fungi. We also analyzed the chemical composition of EOs with the strongest antifungal activity by gas chromatography-mass spectrometry. In addition, we evaluated the antifungal activity of the major active constituents in the selected EOs and elucidated the relationship between the active constituents and their antifungal properties. Finally, we investigated the changes in plasma membrane permeability of T. hirsuta and L. sulphureus caused by selected EOs. 2. Materials And Methods 2.1. Wood decay fungi Trametes hirsuta (CFCC 84683) and Laetiporus sulphureus (CFCC 86368) were procured from China Forestry Culture Collection Center. 2.2. Essential oils and chemicals The antifungal activities of 37 EOs against wood rot fungi in vitro were screened (Table 1). Litsea cubeba and Verbena ocinalis were procured from Rihua Chemical Co. Ltd. (Guangzhou, China). The other EOs was purchased from Huien International Business Co. Ltd. (Shanghai, China). Geraniol, neral, citral, and geranial were purchased from Sigma-Aldrich (China). Page 3/24 Table 1 List of plant essential oils tested for wood-decay fungi oil source of plant family name part origin Chamomile Anthemis nobilis Compositae Flower France Wormwood Artemisia argyi Compositae Leaves China Cypress Cupressus sempervirens Cupressaceae Leaves France Juniper berry Juniperus communis Cupressaceae Fruit France Palmarosa Cymbopogon martini Gramineae Leaves Brazil Citronella Cymbopogon winterianus Gramineae Leaves Java Vetiver Vetiveria zizanoides Gramineae Root India Lavender Lavandula angustifolia Lamiaceae Leaves France Melissa Melissa ocinalis Lamiaceae Leaves France Peppermint Mentha arvensis Lamiaceae Leaves America Basil Ocimum basilicum Lamiaceae Leaves Italy Marjoram Origanum majorana Lamiaceae Flower Egypt Patchouli Pogostemon cablin Lamiaceae Leaves Malaysia Rosemary Rosmarinus ocinalis Lamiaceae Leaves Morocco Clary sage Salvia sclarea Lamiaceae Leaves Russia Litsea Litsea cubeba Lauraceae Fruit China Ravensara Ravensara aromatic Lauraceae Leaves Madagascar Eucalypyus Eucalyptus globulus Myrtaceae Leaves Australia Tea tree Melaleuca alternifolia Myrtaceae Leaves Australia Cajeput Melaleuca leucadendra Myrtaceae Leaves Australia Niaouli Melaleuca viridiora Myrtaceae Leaves Australia Cedarwood Cedrus atlantica Pinaceae Bark America Lignum cedrium Cedrus deodara Pinaceae Heart wood America Black pepper Piper nigrum Piperaceae Fruit India Bergamot Citrus aurantium bergamia Rutaceae Peel Italy Neroli Citrus aurantium amara Rutaceae Flower Egypt Orange Citrus aurantium dulcis Rutaceae Peel Italy Grapefruit Citrus grandis Rutaceae Peel Italy Page 4/24 oil source of plant family name part origin Lemon Citrus limon Rutaceae Peel Italy Mandarin Citrus reticulate Rutaceae Peel Italy Dill Seed Anethum graveolens Umbelliferae Seed China Coriander Coriandrum sativum Umbelliferae Fruit China Caraway Carum carvi Umbelliferae Seed China Fennel Foeniculum vulgre Umbelliferae Seeds Hungary Chuanqiong Ligusticum chuanxiong Umbelliferae Root China Verbena Verbena ocinalis Verbenaceaex Leaves Spain Ginger Zingiber ocinale Zingiberaceae Stem China 2.3. GC-MS The chemical analysis of EOs compounds was determined by GC-MS using an Agilent 6890A/5975C, equipped with a HP-5 capillary column. Analytical conditions were as follows: The GC oven temperature was set at 50°C for 10 min, and raised to 280°C at 10°C/min; He was the carrier gas at 1.0 ml/min; the injection of 1.0 µl; and split ratio of 1:50. The chemical components were identied by NIST mass spectrometry Library (NIST 11.0) and retention index (RI). The relative indices were determined in relation to the series of n-alkanes, with respect to those reported in the literature (Adams, 2007). 2.4. Antifungal assay The antifungal activity of EOs and active compounds were examined using an in vitro assay by Xie et al. (2017a). Briey, 25–400 µg/ml of EOs or their major constituent were added to 20 ml sterilize PDA medium and poured into in 9 cm petri dishes, then inoculation mycelial disc (5 mm) were pace in the center of each dish and incubated at 26 ± 1°C for 5–7 days. Three replicates were done for each dose. When the mycelia reached the edge of control plates (only distilled water), antifungal indices were calculated. 2.5 Membrane integrity determination 2.5.1 Effect of EOs on fungal membrane integrity with propidium iodide (PI) dyeing Membrane integrity of T. hirsuta and L. sulphureus was determined following Yan et al. (2020) method with a confocal laser scanning microscope (CLSM). The fungi were incubated for 24 h in PDB containing the four EOs and its major constituents (1 µl/ml), respectively, and then collected mycelia and stained with PI (1 µg/ml) for 30 min at 28°C in dark. After staining, the mycelia were washed three times with the phosphate buffered saline (PBS, PH 7.4) to remove residual dye. Then use CLSM (Zeiss LSM880, Page 5/24 Germany) to observe PI with excitation/emission wavelengths 561 nm/591 to 635 nm. Each experiment was repeated three times. 2.5.2 Effect of EOs on leakage of fungal nucleic acid and protein Fungal nucleic acid and protein leakage were measured according to the methods of Shao et al. (2013) with slight modications. Fungi were incubated for 24 h in PDB with four EOs or their major constituent (1 µl/ml), then supernatant was used for nucleic